The MBO2/FAP58 heterodimer stabilizes assembly of inner arm dynein b and reveals axoneme asymmetries involved in ciliary waveform

Cilia generate three-dimensional waveforms required for cell motility and transport of fluid, mucus, and particles over the cell surface. This movement is driven by multiple dynein motors attached to nine outer doublet microtubules that form the axoneme. The outer and inner arm dyneins are organized into 96-nm repeats tandemly arrayed along the length of the doublets. Motility is regulated in part by projections from the two central pair microtubules that contact radial spokes located near the base of the inner dynein arms in each repeat. Although much is known about the structures and protein complexes within the axoneme, many questions remain about the regulatory mechanisms that allow the cilia to modify their waveforms in response to internal or external stimuli. Here, we used Chlamydomonas mbo (move backwards only) mutants with altered waveforms to identify at least two conserved proteins, MBO2/CCDC146 and FAP58/CCDC147, that form part of a L-shaped structure that varies between doublet microtubules. Comparative proteomics identified additional missing proteins that are altered in other motility mutants, revealing overlapping protein defects. Cryo-electron tomography and epitope tagging revealed that the L-shaped, MBO2/FAP58 structure interconnects inner dynein arms with multiple regulatory complexes, consistent with its function in modifying the ciliary waveform.


INTRODUCTION
Eukaryotic cilia and flagella are microtubule-based extensions of the cell surface important for cell motility and signaling.Defects in cilia or flagella have been linked to a complex group of diseases collectively termed ciliopathies (reviewed in Reiter and Leroux, 2017).In vertebrate embryos, motility is required for the rotary movement of nodal cilia that determines the left-right body axis (reviewed in Babu and Roy, 2013).In adults, motility is essential for the circulation of cerebrospinal fluid, the clearance of particles and mucus in the respiratory tract, and sperm motility.Defects in the assembly, beating, or signaling of motile cilia have been linked to situs inversus, hydrocephalus, curvature of the spine, chronic respiratory disease, and infertility (Mitchison and Valente, 2017).Given the complex structure of cilia and the large number of genes involved in their assembly and motility, determining the underlying basis for disease can be challenging.The study of model organisms with motile cilia like Chlamydomonas and Tetrahymena has proven useful for understanding the network of genes and proteins required for motility.
Comparative genomics and proteomics have identified >1000 proteins as structural components of the ciliary axoneme and ciliary membrane (reviewed in van Dam et al., 2019;Sakoto-Antoku and King, 2022).Most motile cilia contain nine outer doublet microtubules (DMTs) surrounding two singlet central pair (CP) MTs.Each DMT contains thousands of large, multisubunit motors known as the outer and inner dynein arms (ODA, IDA).The dynein motors generate the force for microtubule sliding between the A-tubule of one DMT and the B-tubule of the adjacent DMT.The ODAs and IDAs are composed of different heavy, intermediate, and light chain (DHC, IC, LC) subunits attached in two rows on the A-tubule.They are further organized into functional units that repeat every 96 nm, with four identical ODAs and seven unique IDAs (I1/f,a,b,c,e,g,d) bound at discrete sites within the repeat (Figure 1).Motor activity is coordinated in part by signals from the CP and its numerous projections that contact three radial spokes (RS1, RS2, RS3, or RS3S) bound near the base of the IDAs in each 96-nm repeat (reviewed in Brown et al., 2023;Witman and Mitchell, 2023).The IC/LC complex of the I1/f dynein forms a regulatory node at the base of RS1, the nexin-dynein regulatory complex (N-DRC) forms another at the base of the RS2, and a calmodulin spoke complex (CSC) connects between the bases of RS2, RS3/RS3S and the N-DRC (Heuser et al., 2009;2012a,b).The I1 dynein and N-DRC also connect to the ODAs and other structures in the 96-nm repeat to stabilize the binding of the IDAs and coordinate dynein activity (Heuser et al., 2009(Heuser et al., , 2012a,b),b); Oda et al., 2013).This network of regulatory proteins includes the tether/tether head (T/TH) structures associated with the I1 dynein motor domains (Fu et al., 2018;Kubo et al., 2018), the MIA complex located between the I1 dynein and the ODAs (King and Dutcher, 1997;Yamamoto et al., 2013), and the FAP57 complex linking the MIA complex, IDAs g and d, and other structures (King et al., 1994;Lin et al., 2019;Bustamante-Marin et al., 2020;Ghanaeian et al., 2023).
Many mutations that disrupt the assembly of ODAs, IDAs, RSs, CP, and its projections, MIA complex, N-DRC, T/TH, and FAP57 complex have been characterized over decades of work in Chlamydomonas, Tetrahymena, and other organisms, and their motility phenotypes are very diverse, ranging from flagellar paralysis, ciliary twitching, reduced beat frequency, and/or altered ciliary waveforms with reduced amplitudes (reviewed in Sale and Dutcher, 2023).However, ciliary motility is extremely complex, and many organisms can adjust the pattern of their waveforms in response to internal and external stimuli.For instance, Chlamydomonas can adjust the beat frequency of its cis and trans cilia to undergo both positive and negative phototaxis, or it can reverse its swimming direction in response to bright light (reviewed in Witman, 1993;Dutcher, 2020;Sale and Dutcher, 2023).The latter involves the conversion of an asymmetric ciliary-type waveform to a more symmetric, flagellar-type waveform.The structures and polypeptides responsible for waveform conversion are poorly understood.A handful of mutations result in cells that swim forward with a symmetric waveform (pf10, pf12/pacrg, fap20; McVittie, 1972;Dutcher et al., 1988;Frey et al., 1997;Dymek et al., 2019), and a second group result in cells that swim backwards with a symmetric waveform (mbo1, mbo2, mbo3; Segal et al., 1984; Table 1).Characterization of fap20 and pf12/pacrg mutants by conventional transmission electron microscopy (TEM) and cryo-electron tomography (cryo-ET) has shown that FAP20 and PACRG form the inner junction between the A-and B-tubules of the DMT (Yanagisawa et al., 2014;Dymek et al., 2019; Figure 1).These studies also revealed secondary defects in the assembly of structures located inside the lumen of the proximal B-tubule (beak-MIP), IDA b, and the I1 dynein T/TH complex (Yanagisawa et al., 2014;Dymek et al., 2019).How defects in the inner DMT junction might lead to changes in waveform asymmetry remains unclear, but the structural defects suggested that fap20 and pacrg mutants may have additional polypeptide deficiencies.Previous studies of mbo1, mbo2, and mbo3 mutants described defects in the assembly of eight axonemal polypeptides ranging in size from ∼33-245 kD, but the identities and locations of the missing proteins were unknown (Segal et al., 1984).The only structural defect identified by conventional TEM was the absence of beak structures located within the lumens of the B-tubules of DMT5 and DMT6.The isolation and characterization of new alleles of MBO2 by insertional mutagenesis later revealed MBO2 to be a conserved, coiled-coil protein found along the length of the axoneme (Tam andLefebvre, 1993, 2002), but the MBO1 and MBO3 gene products remain unknown (Table 1) Here, we reanalyzed the mbo1 and mbo2 mutants to identify the polypeptides and structures responsible for waveform conversion and backwards swimming.We used proteomic approaches and tagged MBO2 transgenes to identify a large group of polypeptides missing or reduced in the mbo2 mutant and restored in MBO2 rescued strains.Comparative proteomics revealed that several proteins reduced in mbo2 were also altered in pf12(pacrg), pf12; fap20 and ida8 (fap57), three other mutants with defects in waveform asymmetry (Table 1).Cryo-ET of mbo2 revealed several DMT-specific structural defects, including changes in an elongated, L-shaped structure reaching from the base of IDA b and extending to other axonemal structures beyond the N-DRC (Lin et al., 2019;Walton et al., 2023).Rescue with SNAP-tagged MBO2 constructs followed by streptavidin gold labeling and classification averaging shows that MBO2 is one of the components of the L-shaped structure that interconnects multiple regulatory components and stabilizes the assembly of IDA b within the 96-nm axoneme repeat.

Identification of polypeptide defects in mbo2 and mbo1 axonemes
Transformation of mbo2 with an HA-tagged MBO2 transgene rescued the mbo motility phenotype, increased forward swimming velocities, and restored assembly of the missing MBO2 subunit along the length of the axoneme (Figure 2, A-C; Supplemental Figure S1; Supplemental Tables S2 and S3, see also Tam and Lefebvre, 2002).The forward swimming velocities observed in four independent HAtagged transformants were not completely wild-type (Figure 2B, consistent with previous studies [Tam and Lefebvre, 2002]).To identify polypeptides associated with MBO2, we purified axonemes from wild-type (WT), mbo2, MBO2-HA rescued strains, and mbo1, labeled them using iTRAQ isobaric tags, and subjected them to quantitative mass spectrometry (see Materials and Methods).Although the MBO1 gene product has not been identified, earlier work showed that mbo1 and mbo2 mutants have similar deficien-  (Bt).The inner junction between the At and Bt is composed of alternating subunits of PACRG and FAP20 (aqua).The A-tubule contains a multisubunit ODA with three motor domains (lilac), multiple IDA isoforms (pink), the NDRC (yellow), and one of three radial spokes (RS, grey).The small light blue dot indicates the location of the ruler subunits FAP59 (CCDC39) and FAP172 (CCDC40) that determine the spacing of the RSs and IDAs.Also shown in cross-section is a portion of the FAP57 complex (dark blue).(C) Diagram of a single DMT shown in longitudinal view from the proximal (pro) to distal (dist) region of the 96-nm repeat, containing four ODAs (lilac), the seven IDA isoforms I1 (f), a, b, c, e, g, d (pink), and the three RS structures (RS1, RS2, RS3S), with I1 (f) above RS1 and the N-DRC (yellow) above RS2.The two I1 motor domains (α, β) are also attached to the DMT through the Tether-Tether Head (T/TH) (fuschia).The MIA complex (brown) links the base of I1 to the ODAs, the DMT, and the proximal portion of the FAP57 complex (dark blue).The coiled-coil domains of the FAP57 extend beyond the N-DRC to contact IDA g and d.
cies in axonemal polypeptides (Segal et al., 1984, see Table 1).The abundance of each polypeptide present in mbo mutant axonemes was expressed as a protein ratio relative to its abundance in WT axonemes.Nineteen proteins were significantly reduced below 0.75 (P value < 0.05) in four iTRAQ experiments using mbo2 and mbo1 (Supplemental Table S4).We later modified the protocol using three independent samples each of WT, mbo2, and MBO2-HA axonemes, TMT-based isobaric tags, and more sensitive instrumentation (Supplemental Figure S2A).Thirty-eight polypeptides were identified as significantly reduced (P value < 0.05) in mbo2 axonemes relative to the MBO2-HA rescued samples (Supplemental Table S4).Fourteen of these were detected by a small number of peptides, showed limited sequence coverage, and were poorly represented in previously published proteomes of WT axonemes; many were also poorly conserved outside of green algae.Their characteristics are listed in Supplemental Table S4 and are not discussed further here.The remaining 24 proteins were detected by several peptides, showed broad sequence coverage, and were well represented in previous proteomes (Table 2, Supplemental Table S4).Their characteristics are summarized in Table 3.
One unexpected finding was the reduction of DHC5 in mbo2 axonemes.DHC5 is one of 12 DHCs forming the inner dynein arms (IDA) in Chlamydomonas (reviewed in King et al., 2023).To confirm the decrease of DHC5, we probed Western blots of WT and mutant axonemes with DHC5 and DHC9 specific antibodies.DHC5 was reduced in mbo2 and restored to WT levels in MBO2-HA rescued axonemes, whereas DHC9 was present at WT levels in all samples (Figure 2C).Proteomic analysis indicated no significant changes in any of the other DHCs in mbo2 axonemes compared with WT (Supplemental Table S4).Although the mutant gene products of mbo1 and mbo3 remain uncharacterized, both MBO2 and DHC5 were reduced in mbo1 and mbo3 axonemes (Figure 2D; Supplemental Table S4).These observations suggest that MBO2 and its associated proteins may stabilize assembly of DHC5 onto the DMT.To better understand the relationship between DHC5 and MBO2, we tested whether the two polypeptides might cofractionate following biochemical extraction of purified axonemes.The IDAs can be extracted from WT axonemes by treatment with 0.6 M NaCl or KCl (Pazour et al., 1995), but extraction of MBO2 required treatment with 0.4-0.6 M NaI or 0.5% Sarkosyl (Figure 2, E and F), indicating that they are in distinct biochemical complexes.A subset of the proteins reduced in mbo2 are enriched in 0.6M NaCl/0.6MKCl extracts of WT axonemes (Table 3; Supplemental Table S4; Pazour et al., 2005), suggesting that they might be associated with DHC5.The IDAs in 0.6 M NaCl extracts can be partially fractionated by ion exchange FPLC based chromatography into seven distinct peaks containing different dynein isoforms known as IDA a-g (Kagami and Kamiya, 1992).DHC5 is the HC subunit of IDA b (Yagi et al., 2009).Proteomic analysis of the FPLC peak containing IDA b and DHC5 identified several polypeptides, but only one protein, Cre14.g618300, was also reduced in mbo2 (Table 2; Supplemental Figure S2B; Supplemental Table S4).These results indicate that most of the  MBO2-associated proteins dissociate from DHC5 during high salt extraction and FPLC fractionation and therefore do not qualify as bona fide subunits of the IDA b complex.However, at least some of proteins reduced in mbo2 may stabilize binding of IDA b to the DMT, similar to the way that subunits of the N-DRC stabilize the binding of IDA e and g (Bower et al., 2013).

Comparative proteomics of other mutants with phenotypes similar to mbo2
The pf12/pacrg and pf12; fap20 mutants swim forwards with a symmetric waveform, lack beaks inside the B-tubules of DMTs 5 and 6, and assemble reduced levels of IDA b into the axoneme (McVittie, 1972;Frey et al., 1997;Dymek et al., 2019, see Table 1).Given these similarities in phenotypes with mbo strains, we analyzed the proteomes of pf12 and pf12; fap20 axonemes to look for potential overlap with the proteins missing in mbo1 and mbo2.The pf12 mutation is a null allele of the PACRG gene, which encodes one of two proteins that form the inner junction between the A-and B-tubules of the DMT (Dymek et al., 2019; Figure 1).FAP20 is the second subunit of the inner DMT junction (Yanagisawa et al., 2014).Proteomics of pf12 axonemes (using iTRAQ labeling) and pf12; fap20 axonemes (using TMT labeling) confirmed that PACRG is reduced to < 10% of WT levels, whereas MBO2 is present at WT levels, as also shown by Western blot (Supplemental Table S4; Figure 2G; Dymek et al., 2019).However, both pf12 and pf12; fap20 showed significant reductions in other axonemal proteins, including several proteins that were reduced in mbo2 (Tables 1-3; Supplemental Table S4).
The shared proteins may include potential docking factors for IDA b/DHC5, beak components, or subunits of other complexes destabilized in mbo, pacrg, and fap20 mutants (Tables 1-3).
The mbo mutants also have defects in assembly of a subset of proteins recently associated with the FAP57 complex (Tables 1-3; Supplemental Table S4; Lin et al., 2019).FAP57 is a conserved, WD repeat, coiled-coil protein that extends from the MIA complex to beyond the distal end of the 96-nm repeat (Figure 1).Null mutations in FAP57 (bop2, ida8, fap57) have a slow swimming phenotype, partially suppress the symmetric waveforms of pf10 mutants, and disrupt assembly of FAP57, an EF-hand, WD repeat protein known as FAP337, and three IDA HCs (Dutcher et al., 1988;Lin et al., 2019;Bustamante-Marin et al., 2020;Table 1;Supplemental Table 4).The loss of FAP57 in these mutants is partially offset by increases in the assembly of two paralogues, FBB7 and FAP331.Likewise, the decrease in FAP337 is partially offset by increases in its paralogue, Cre07.g313830(Lin et al., 2019;Bustamante-Marin et al., 2020; Table 1; Supplemental Table 4).We, therefore, compared the proteomes of mbo2, pf12, pf12; fap20, and ida8 axonemes to determine the extent of overlap between the different polypeptide defects.
As described in Supplemental Table 4, the FAP57 paralogue FAP331 was reduced in axonemes from mbo, pf12, and pf12; fap20 strains, FAP57 levels were WT in all mutants, and FBB7 was variably reduced in pf12 and pf12; fap20.Likewise, the two EF-hand, WD repeat proteins, FAP337 and Cre07.g313850, were reduced with variable degrees of significance in the different mutants (Supplemental Table S4; Table 3).However, none of the other polypeptides reduced in mbo2 were significantly altered in the ida8/fap57 axonemes (Supplemental Table S4).These results suggest that fluctuations in assembly of the FAP57 paralogues and their associated subunits are secondary defects due to the absence of other proteins in mbo, pf12, and pf12; fap20 axonemes (see Discussion).
We recently analyzed the proteome of a pf9-2; pf28 double mutant strain with short flagella (∼2.9 μm) using iTRAQ labeling and MS/MS to identify proteins that might be enriched in the proximal or distal regions of the axoneme (Hwang et al., 2024).DHC5 is reduced in the proximal 2 μm of WT flagella and reduced in short flagella during regeneration (Yagi et al., 2009).Proteomic analysis of short pf9-2; pf28 axonemes confirmed that the proximal DHCs (DHC3, DHC4, DHC11) were increased > twofold (see Table 2 in Hwang et al., 2024).Here, we examine the levels of the MBO2-associated proteins and confirmed that DHC5 was significantly reduced   (∼0.29) in short axonemes relative to WT axonemes (Table 3; Supplemental Table S4).Interestingly, the MBO2-associated proteins FAP324, FAP343, and FAP238, were also reduced below 0.75 in short axonemes (Supplemental Table S4); these proteins were also found in high salt extracts and may potentially interact with DHC5 (Table 3).Three MBO2-associated proteins were increased in pf9-2; pf28 axonemes (mutant/WT ratio > 1.4), and all were poorly conserved outside green algae (Supplemental Table S4; Table 3).These proteins may be associated with structures located in the proximal region.However, most MBO2-associated proteins were found at WT levels in pf9-2; pf28 axonemes (Supplemental Table S4).Most FAP57-associated proteins were found at WT levels in short flagella, but both FAP331 and FAP337 were reduced (Supplemental Table S4).Thus, most MBO2-associated and FAP57-associated proteins are found along the length of the axoneme, but a subset may vary between the proximal and distal axoneme.or change swimming direction.The IO mutant (CC-2012) is an isolate of pf23 that assembles full-length, paralyzed flagella lacking most of the IDAs, including IDA b/DHC5.However, Western blots showed that MBO2 levels were WT (Figure 2G).We also searched the CLiP library of insertional mutants (Li et al., 2016;2019) to identify potential dhc5 mutations (Supplemental Table S1).PCR of genomic DNA identified four strains with plasmid insertions into exons located in the 5′ region of the DHC5 gene (Supplemental Figure S3, A-C).Western blots of axonemes probed with a DHC5 antibody detected bands migrating at the sizes predicted for N-terminal fragments containing the tail domain but lacking the C-terminal motor domain.MBO2 was also present at WT levels (Supplemental Figure S3D).Analysis of swimming phenotypes showed that the dhc5 strains swam forwards at WT speeds with no obvious motility defects (Supplemental Figure S3E), demonstrating that absence of the DHC5 motor domain does not result in a mbo phenotype.

Cryo-electron tomography reveals defects in assembly of IDA b and several DMT-specific structures
Studies of mbo axonemes by conventional TEM have shown defects in the assembly of beak structures within the lumens of the B-tubules of DMT5 and DMT6 but failed to reveal any defects in the assembly of the IDAs (Segal et al., 1984;Tam and Lefebvre, 2002).These earlier studies analyzed random cross-sections of fixed axonemes and did not employ any image averaging.We used cryo-ET, computational averaging of the 96-nm repeat, and classification analysis to reanalyze various structures, including the IDAs, in WT and mbo2 axonemes (Figure 3).Sub-tomogram averages of 96-nm repeats from all WT DMTs revealed the two-headed I1/f dynein at the proximal end of the repeat, followed by the six, single-headed IDAs (a, b, c, e, g, d; Figure 3, A-D).However, the electron density of IDA b, which contains DHC5 and is located just distal to RS1, was weaker than for other IDAs, suggesting IDA b was not present in all WT 96-nm repeats (Figure 3, A-D).In contrast to WT, tomograms of the 96-nm repeat from all DMTs in mbo2 lack IDA b almost entirely (Figure 3, E-H).As previously reported for WT axonemes, IDA b is significantly reduced on all DMTs in the proximal region of the axoneme (Yagi et al., 2009) and specifically reduced on certain DMTs in the medial/distal region (Bui et al., 2012;Lin et al., 2012Lin et al., , 2019;;Dymek et al., 2019).Therefore, we performed classification analyses to determine the presence or absence of IDA b in different regions of the axoneme and on specific DMTs (Figure 3, I and J).In WT, IDA b was present in 6% of the proximal tomograms and 55% of the medial/distal tomograms; in mbo2, IDA b was only present in 2% of the proximal tomograms and 12% of the medial/distal tomograms (Figure 3I).In WT axonemes, IDA b was reduced on all DMTs in the proximal region and on DMT1, DMT5, and DMT9 in the medial/distal region (Figure 3J), consistent with previous reports (Bui et al., 2012, Lin et al., 2012, 2019;Dymek et al., 2019).In mbo2, IDA b was reduced in all DMTs, with the most significant changes from WT in the medial/distal region of DMT2 to DMT8 (Figure 3J).
Examples of the distribution of IDA b in individual axonemes are shown in Figure 3K.
Because several polypeptides are missing or reduced in mbo2 axonemes, we analyzed the region around IDA b using DMT-specific averaging (Figure 4).The averages of DMT1 and DMT9 did not show significant changes between WT and mbo2 (Figure 4, C and  K).These results are consistent with the observation that IDA b was rarely found on these DMTs in either WT or mbo2 (Figure 3J).For DMT5 to DMT8, both IDA b and the WT structures near the base of IDA b were completely missing in mbo2 (Figure 4, G-J).This suggests that a subset of the proteins missing in mbo2 are located within the structures around the base of IDA b.In contrast, the mbo2 defects were different in the averages of DMT2 to DMT4.Even though IDA b was missing in mbo2, the region surrounding the base of IDA b retained significant structural density, similar but not identical to the structures seen in WT (compare purple/blue density in Figure 4, D-F).These structures may contain other proteins that are related to proteins missing in mbo2, but that do not depend on MBO2 for assembly into the axoneme (see Discussion).
Tomograms from the proximal region of the axoneme were also analyzed for the presence or absence of the beak structures inside the B-tubules of DMT1, DMT5, and DMT6 (Figure 5).Beak structures were visible in DMT1 and DMT6 from both WT and mbo2 axonemes (Figure 5, A, B, E, and F) but not visible in DMT5 from mbo2 axonemes (Supplemental Figure 5, C and D).DMT-specific averaging also revealed several other densities associated with the MBO2 complex that varied between DMTs in WT axonemes; several examples are shown in Figure 5, G-J.

Rescue of mbo2 defects and mapping the position of MBO2 by transformation with SNAP-tagged MBO2 transgenes
The large number of MBO2-associated proteins and the complexity of structural defects in mbo2 axonemes make precise localization of the MBO2 subunit impossible using a WT-mutant comparison.Therefore, we constructed three MBO2 transgenes with SNAP tags located at the N-terminus (N-SNAP), near the middle of the protein (M-SNAP at amino acid 569) and at the C-terminus (C-SNAP; Figure 6A; Supplemental Figure S1).Each transgene was introduced into mbo2 by cotransformation, and transformants were screened for recovery of forward swimming and reassembly of MBO2 and DHC5 into the axoneme.Western blots of axonemes from rescued strains demonstrated that MBO2 was assembled at WT levels and migrated at the size expected for a SNAP-tagged subunit.Assembly of DHC5 was also restored in the rescued strains (Figure 6B).Measurement of swimming velocities indicated the N-SNAP and C-SNAP rescued strains recovered ∼72% of WT velocity, whereas the M-SNAP rescued strain recovered only ∼48% of the WT speed (Figure 6C).However, analysis of cell trajectories and motility by phase contrast microscopy confirmed that all the rescued strains were swimming forwards with asymmetric waveforms (Figure 6D).The SNAP-tagged transgenes therefore restored sufficient function to rescue the mbo phenotype, even though the rescued cells were not completely WT with respect to swimming velocities.
To obtain a more quantitative measurement of recovery, we analyzed the DHC composition of the rescued strains by SDS-PAGE, Three independent samples (biological replicates) of WT, mbo2-4, and MBO2-HA axonemes were labeled with three separate TMT reagents (nine samples total) and analyzed by mass spectrometry.The Chlamydomonas genome ID number is shown in the first column, and the common name of the protein is shown in the second.The predicted molecular weight is shown in the third column and the percent coverage of the protein sequence is shown in the fourth.The number of unique peptides used for protein identification is shown in the fifth column.The mbo2/WT ratios are shown sixth column, and mbo2/MBO2-HA ratios are shown in the seventh.Ratios that were significantly different from control samples (P < 0.05) are shown in bold.The increase or decrease in protein ratios in other mutant axonemes are noted in the last column.See Supplemental Table S4 for additional details on other proteins identified during proteomic analyses of mbo2 and other mutants (ida8/fap57, pf12/pacrg, pf12; fap20, short pf28 pf9-2).mass spectrometry, and cryo-ET.Axonemes from WT, mbo2-4, and the three SNAP-tagged rescued strains were fractionated by SDS-PAGE, and the DHC region was excised for MS/MS analysis (see boxed region in Supplemental Figure S2C).The relative abundance of each IDA DHC was estimated by spectral counting and plotted as a percentage of DHC content of the I1 dynein (Supplemental Figure S2D).Quantification of peptides using the tools available in Proteome Discover yielded similar results (Supplemental Table S4).
Only DHC5 was significantly reduced in mbo2, and it was reassembled in the SNAP-tagged rescued axonemes at 64-84% of WT levels.Inspection of tomograms from the medial and distal regions of axonemes confirmed that IDA b was present in 14% of the 96-nm repeats in mbo2 and increased to 35-49% of the repeats in the SNAP-tagged rescued strains, compared with 61% of the repeats in WT (Figure 6E).Analysis of individual DMTs showed that recovery of IDA b in the SNAP-tagged strains could be observed on DMT2 to DMT8, with the C-SNAP closest to WT and M-SNAP showing more DMT-specific variability, reaching almost WT levels on DMTs 6 and 7, but significantly less on the remaining DMTs (Figure 6F).
To determine how the MBO2 polypeptide might be arranged relative to the structures associated with IDA b, we treated axonemes with biotin-streptavidin gold (∼1.4 nm) to label the SNAP tags for in situ visualization by cryo-ET.Control experiments using silver enhancement procedures to stain samples on gels (Song et al., 2015) confirmed that the SNAP-tags were accessible to streptavidin-gold (Figure 6G).To identify additional densities, that is, density not visible in WT averages and thus corresponding to the streptavidingold labels, we classified the subtomogram volumes of the 96-nm repeats, revealing the tag-density at distinct locations within the repeat (Figures 6H and 7).Specifically, in the N-SNAP rescued strain, the recovery of IDA b was evident in 41% of the averaged repeats compared with 61% in WT (Figure 6E), and an additional label-density was observed close to the IDA b tail domain in ∼20% of the repeats (compare white and yellow arrowheads in Figure 7, A and B).In the M-SNAP strain, 49% of the repeats contained IDA b (Figure 6E), and an additional label-density was observed near the distal end of the MBO2-associated complex in ∼15% of the repeats (yellow in Figure 7D).In the C-SNAP strain, 35% of the repeats contained IDA b (Figure 6E), and an additional label-density was observed close to the IDA b tail and inner junction region in ∼31% of the repeats (yellow in Figure 7F).This suggests that both the N-terminus and C-terminus of MBO2 are located between RS1 and RS2, in the vicinity of the IDA b attachment site, possibly stretching a full 96-nm repeat as a L-shaped structure (see Discussion).
We did not detect additional densities for streptavidin gold in all repeats of the SNAP-rescues, even though we observed significant recovery of IDA b and MBO2-associated structures in all three SNAP-tagged strains (Figure 6, E and F).Given the differences between DMTs in the mbo2 mutant, we considered two possibilities.The first is that the accessibility of the SNAP-tags in the MBO2 The proteins and Chlamydomonas genome ID numbers are listed in the first column.The total number of amino acids and predicted molecular weight (in parenthesis) are noted in the second column.The predicted protein domains were identified by the SMART program: AAA (ATPase), CC (coiled-coil), EF-hand (calcium binding motif), FN3 (fibronectin-like domain), IQ (calmodulin binding domain), Kelch (beta strand-propeller domain), WD (WD repeat), WW (WW repeat).The predicted amino sequences were compared against the Volvox carteri, Physcomitrella patens, and the human (Homo sapiens) genomes; the names (in bold) and accession numbers of the best hits are shown, along with the BlastP or PHI-Blast score (in parenthesis).ND, not detected or not determined.The proteins reduced in mbo2 were assorted into groups based on changes in their protein ratios in different mutant axonemes, their susceptibility to extraction with 0.6 M KCl (Pazour et al., 2005), and their relative abundance in WT axonemes (Sakato-Antoku and King, 2022; see Supplemental Table S4).subunit to the streptavidin-gold particles might vary within the complex of associated proteins in the axoneme.The second is that our ability to detect the SNAP tags might vary between different DMTs.We, therefore, checked the tomograms to determine which DMTs were associated with the label densities (Figure 6H).We found that the C-terminal tag was detected mostly on DMT2 to DMT8.However, the N-terminal and M-SNAP tags were detected more frequently on DMT6 to DMT8 than other DMTs.These observations suggest that the MBO2 subunit is located on DMT2 to DMT8, but the N-and M-SNAP tags are more accessible to gold labeling on DMT6 to DMT8.

Characteristics of proteins missing or reduced in mbo axonemes and their potential interactions
MBO2 is a conserved, coiled-coil protein that is tightly bound along the length of the axoneme, and two-dimensional-PAGE previously indicated that mbo mutants have defects in the assembly of at least eight unidentified axonemal proteins (∼33-245 kD; Segal et al., 1984;Tam and Lefebvre, 2002).We used five rounds of isobaric labeling (iTRAQ or TMT) and quantitative MS/MS to identify multiple proteins that were missing or reduced in mbo2 axonemes and restored in MBO2-HA rescued axonemes (Table 2; Supplemental Table S4).Here we discuss the 24 most abundant polypeptides that were significantly reduced in the TMT experiment as potential candidates to be part of a MBO2 complex (Tables 2 and 3).To gain further insight into these proteins, we analyzed their susceptibility to extraction with high salt (0.6M NaCl/KCl), their cofractionation with IDAs by FPLC, and their relative abundance in other motility mutants that alter waveform asymmetry or ciliary length.The proteins were sorted into five groups whose characteristics are summarized in Table 3.The first group contains MBO2 and four other proteins, FAP58, Cre06.g289600,Cre14.g618300, and FAP145, that may coassemble with MBO2 as part of the L-shaped structure in the 96-nm axoneme repeat.All four proteins are relatively abundant, coiled-coil proteins, significantly reduced only in mbo2, but not altered in pf12, pf12; fap20, or short flagella mutants (Tables 1-3; Supplemental Table S4; Pazour et al., 2005;Lin et al., 2019;Sakoto-Antoku and King, 2022).Cre06.g289600 is not highly conserved, but MBO2 and FAP58 share significant sequence homology (33.5% identity, Blast P score 3e-44) and even greater homology to different proteins in other species with motile cilia and flagella (Nevers et al., 2017).FAP58 is also closely related (∼80% identity) to a Chlamydomonas paralogue, FAP189.FAP58, however, is reduced in all mutants with a mbo phenotype, whereas FAP189 is unchanged or possibly elevated in mbo strains (Tables 2 and 3; Supplemental Table S4).Thus, the FAP58 and FAP189 paralogues may play distinct but complementary functions in Chlamydomonas.A recent cryo-EM, single particle analysis visualized parts of the L-shaped structure on the A-tubule with higher resolution, revealing a coiled-coil filament.The authors proposed a pseudoatomic model, placing a FAP189 homodimer within the L-shaped coiled-coil structure on the surface of the DMT (Walton et al., 2023).We propose that MBO2 and FAP58 form a coiled-coil heterodimer that complements the role of a FAP189 homodimer on different DMTs or regions.Recent chemical crosslinking and proteomic analysis of the MBO2 and FAP58 orthologs in Tetrahymena indicate that these two subunits interact closely throughout their lengths in the Tetrahymena axoneme (McCafferty et al., 2023), consistent with our hypothesis.MBO2 is the orthologue of the verte-brate polypeptide CCDC146 (29.5% identity, Blast P score 1e-103), and ccdc146 mutations result in defective sperm formation and male infertility (Muroňová et al., 2023).Interestingly, there is only a single orthologue of FAP58 in most species (Nevers et al., 2017), known as CFAP58/CCDC147 in vertebrates (∼50% identity).CCDC147 is abundantly expressed in the testis and to a lesser extent in the lung, heart, and endometrium, and ccdc147 mutations are associated with abnormal sperm flagella morphology and rare germline mutations associated with lung cancer (Liu et al., 2016;He et al., 2020;Sha et al., 2021).Whether ccdc146 or ccdc147 mutations alter ciliary waveforms in other species is currently unknown.
Two smaller proteins are also reduced only in mbo2, FAP145 and Cre14.g618300, and both can be extracted in high salt (Table 3; Supplemental Table S4) FAP145 is weakly related to a translin associated factor X interacting protein but is otherwise uncharacterized.Cre14.g618300 was detected in FPLC peak b, along with DHC5.Additional studies are needed to determine whether these proteins might facilitate interactions between the MBO2/FAP58 heterodimer and IDA b.
The second group of proteins reduced in mbo2 includes two proteins previously associated with the FAP57 complex, FAP331 and FAP337 (Lin et al., 2019;Bustamante-Marin et al., 2020).Both are extracted in high salt and altered in ida8/fap57 mutants (Supplemental Table S4; Tables 1-3).The FAP57-associated proteins also fluctuate in mbo1, pf12, and pf12; fap20 strains.These are consistent with observations that the FAP57 complex interacts with multiple structures in the axoneme, including an L-shaped structure (Lin et al., 2019;Ghanaeian et al., 2023;Walton et al., 2023, see below).
To identify other polypeptides that might facilitate interactions between DHC5 and MBO2, we searched for proteins that were enriched in high salt extracts and significantly reduced in short (pf28; pf9-2) axonemes (Supplemental Table S4; Pazour et al., 2005;Hwang et al., 2024).This group contains DHC5 and three other polypeptides, FAP324, FAP238, and FAP343 (Tables 1-3; Supplemental Table S4).FAP324 (∼70 KD) is significantly reduced below 17% in all mbo proteomics experiments and contains an N-terminal  coiled-coil domain and C-terminal Kelch and FN3 domains.It shares limited homology to KLHDC3, which is abundantly expressed in the testis.FAP238 is a small (42 kD), coiled-coil protein with an EF-hand domain that may function as a calcium sensor.FAP343 is weakly related to CEP164.Both FAP238 and FAP343 are also reduced in the pf12 strains (Table 3).
A larger group of proteins were reduced in either pf12 and/or pf12; fap20 but not altered in short axonemes (Table 3; Supplemental Table 4).Cre3.g144887 is a relatively abundant protein that contains an IQ domain and a LRRC domain with limited homology to the LRRC74a protein found in testis.This protein is also present in salt extracts.FAP423/MOT12 is a minor component based on previous proteomes (Pazour et al., 2005, Sakoto-Antoku andKing, 2022), but it contains an IQ motif and is related to a human protein, C11orf65, that is relatively abundant in testis and conserved in other species with motile axonemes (Li et al., 2004).Two other proteins, FAP271 and Cre12.g555378,share limited homology with a sperm-tail PG-repeat domain containing protein also abundant in testis.The remaining six proteins are coiled-coil or low complexity proteins that were reduced in pf12 and/or pf12; fap20 but are poorly conserved outside of green algae.

Cryo-ET of mbo2 reveals multiple DMT-specific defects
Cryo-ET of mbo2 axonemes confirmed the loss of IDA b in the 96-nm repeat (Figure 3), and transformation of mbo2 with tagged MBO2 constructs rescued the mbo motility phenotype and restored assembly of DHC5 and IDA b into the axoneme (Figures 2 and 6).Thus, although MBO2 and DHC5 are members of distinct biochemical subcomplexes, MBO2-associated proteins are required for stabilizing the assembly of DHC5 at a specific site in the 96-nm repeat.To investigate how these complexes might interact within the axoneme, we turned to DMT-specific averaging.Immunofluorescence microscopy first demonstrated that DHC5 is severely reduced in the proximal 2 μm of WT axonemes (Yagi et al., 2009), and cryo-ET later showed that IDA b is also missing or reduced on DMT1, DMT9, and DMT5 in the medial-distal region of WT axonemes (Bui et al., 2012;Lin et al., 2012;2019;Dymek et al., 2019).In mbo2, IDA b is reduced on all DMTs (Figures 3 and 4) but reassembled on DMT2 to DMT8 in MBO2 rescued strains (Figure 6).DMT-specific averaging further revealed that the L-shaped structures located below the tail domain of IDA b are heterogenous in appearance on different DMTs in both WT and mbo2 (Figure 4).More specifically, the L-shaped structure (indicated in red in Figures 4 and 8), located between RS1 and RS2 on DMTs 5 to 8 in WT axonemes is missing on DMTs 5 to 8 in mbo2.A slightly different L-shaped structure is present on DMTs 2 to 4 in both strains (indicated in purple in Figures 4 and  8).The heterogeneity in structures suggests that some of the proteins reduced in mbo2 are located on DMTs 2 to 8, whereas other subunits may be restricted to specific regions of the axoneme or a smaller subset of DMTs.
The mbo, pacrg, and fap20 mutants share defects in the assembly of the B-tubule beak-MIP structures located inside DMTs in the proximal region of the axoneme (Segal et al., 1984;Yanagisawa et al., 2014;Dymek et al., 2019).More specifically, mbo2 lacks beaks in DMT5 (Figure 5), and both pacrg and fap20 display beak defects in DMTs 1, 5, and 6 (Yanagisawa et al., 2014;Dymek et al. 2019).How defects at the IJ of the DMT (in the case of fap20 and pf12) or on the surface of the A-tubule (in the case of mbo2) are both associated with defects in beak structures in the proximal B-tubules and defects in assembly of IDA b along the length of the axoneme is not yet understood.Holes in the IJ of the DMT may alter the stability of MIPs inside the B-tubule and influence the binding of other proteins on the DMT surface.Likewise, some of the proteins missing in the mbo strains may interact directly or indirectly with the DMT junction or MIP proteins.Further characterization of mutations in those proteins that are reduced in both the IJ and mbo mutants (Table 3; Supplemental Table 4) could provide new insights into the overlapping protein network and the phenotypic similarities between the strains (i.e., symmetric waveforms, defective beaks, reduced IDA b).Likewise, further characterization of proteins that are uniquely missing in mbo mutants (Table 3) could provide a better understanding of the mechanism that converts the waveform from forwards to backwards swimming (Table 3).Epitope-tagged MBO2 constructs rescue the mbo phenotype and most of the DHC5/IDA b defects Transformation with tagged MBO2 constructs restores forward swimming, but none of the rescued strains swim at completely WT velocities (Figures 2 and 6;Tam and Lefebvre, 2002).To better understand why swimming velocities might be reduced, we analyzed the reassembly of DHC5/IDA b using multiple proteomic approaches and cryo-ET.In general, the recovery of the swimming velocity could be correlated with the extent of DHC5/IDA b recovery (Figure 6; Supplemental Figure S2; Supplemental Table S4).However, disruption of the DHC5 motor domain by itself had negligible effects on motility (Supplemental Figure S3).These observations suggest that the interaction of IDA b with the MBO2-associated subunits is more critical for regulating motility than the activity of the DHC5 motor domain alone.Interactions within the MBO2 complex may influence coordination with other dyneins in the axoneme and/or alter mechanical properties such as resistance or elasticity.Indeed, interactions between dynein motor and tail domains has been shown to alter motor activity in many systems (McKenney, 2019;Rao et al., 2021).

Localization of the MBO2 SNAP tags within the 96-nm repeat
Given the heterogeneity of structural defects in the mbo2 mutant, we turned to streptavidin gold labeling of SNAP tags, DMT-specific averaging, and class averaging to determine the approximate location of the MBO2 polypeptide.Labeling of the C-terminal SNAP tag and DMT-specific averaging suggested that MBO2 is present on DMT2 -DMT8 (Figure 6H).Classification averaging of all DMTs from C-SNAP tagged axonemes detected an additional density close to the inner junction, below the shorter part of the L-shaped structure that is missing from DMT5 to DMT8 in mbo2 (Figures 7 and 8).The additional density marking the N-terminal SNAP tag is located above and slightly proximal of the C-terminal tag.It sits above the surface of the DMT, close to the tail domain of IDA b, but below the motor domain (Figures 7 and 8).The additional density marking the M-SNAP tag at amino acid 569 was detected beyond RS2, and near the surface of the DMT, between protofilaments A04 and A05 (Figures 7 and 8).This site is close to the distal end of the red structures detected as missing by cryo-ET of the mbo2 mutant.
One possible interpretation of these observations is that the Nterminus of MBO2 is located close to IDA b and the polypeptide extends distally towards RS2; the MBO2 subunit then folds back proximally and turns downward towards the C-terminus, so that the N-and C-termini of one MBO2 subunit are both located near the same IDA b docking site.However, the MBO2 sequence is predicted to form several coiled-coil domains separated by regions whose structure cannot be predicted with high confidence (Figures 2A and 6A;Tam and Lefebvre, 2002).An alternative interpretation consistent with both the locations of the labels and the size and predicted structure of MBO2 polypeptide is that the MBO2 subunit adopts an extended confirmation spanning the 96-nm repeat, like that observed for the CCDC39/CCDC40 (FAP59/ FAP172) subunits (Oda et al., 2014;Gui et al., 2021).We favor this second interpretation based on the evidence described below.

Model for the proposed location of the MBO2 subunit and its interaction with other complexes in the axoneme
We identified a candidate L-shaped structure in our highest resolution images of WT Chlamydomonas axonemes that we propose as the likely location of the MBO2 polypeptide on DMTs 2-8 (Figure 9; Supplemental Table S5).We suggest that the C-terminal region of MBO2 corresponds to a filamentous structure that can be seen close to the surface of the A-tubule in cross-section, with the C-terminus located near protofilament A02 (Figure 9, A and B), consistent with the location of the C-terminal SNAP tag shown in Figure 7.The C-terminal region of the filament wraps around the A-tubule (circled in red/colored red in Figure 9, A and B) to a cleft between protofilaments A04 and A05.Rotating the images 90° along the y-axis (Figure 9, C and D) shows the position of the filament running vertically from protofilament A02 to A04 (red arrowheads in Figure 9D) and then turning distally and running along the surface of the DMT between protofilaments A04 and A05 to the red arrowhead on the right (Figure 9D).This filament is modeled in red in the isosurface rendering (Figure 9C).Tilting the isosurface rendering 60° as shown in Figure 9E, the MBO2-associated, L-shaped structure (red) interacts with the axoneme ruler (gold) at its C-terminus and then extends vertically, running below the base of IDA b (green) to contact the MIA complex (orange).The red structure turns distally to extend along the surface of the DMT, running below the FAP57 complex (blue) and N-DRC (yellow) and beyond the RS3S (unshaded) into the next 96-nm repeat, where it fades just below the I1 dynein, ∼5.5 nm from the predicted location of the N-terminal SNAP tag.
The L-shaped structures present in WT and missing in mbo2 (Figures 4 and 7-9) are similar in location and organization to a Lshaped, coiled-coil structure described in cryo-EM studies of WT axonemes (see Figure 1 in Ma et al., 2019;Extended Data Figure 4 in Gui et al., 2021).This coiled-coil structure has recently been proposed as the location of FAP189 based on limited fitting of short regions of an AlphaFold2 predicted structure for a FAP189 homodimer (predicted in several sequence fragments) to a single particle, cryo-EM reconstruction of WT axonemes (Extended Data Figures 5 and 8 in Walton et al., 2023).However, we have found that the decrease in MBO2 seen in all the mbo mutants is more closely correlated with a decrease in FAP58, whereas FAP189 levels are essentially unchanged or even increased in mbo mutant axonemes (Table 2; Supplemental Tables S4).This suggests that at least on DMTs 5-8, where the L-shaped structure is missing in mbo2, MBO2 and FAP58 form a heterodimer contained within the L-shaped, coiled-coil filament and that they also serve as a scaffold for the assembly of other polypeptides missing in mbo axonemes.We further hypothesize that the MBO2/FAP58 heterodimer and FAP189 homodimer perform distinct but complementary functions in stabilizing the binding of other proteins in the 96-nm repeat.MBO2/FAP58 and FAP189 may occupy similar sites on different DMTs or vary in proximal, medial, or distal regions.For instance, a FAP189 homodimer may form part of the L-shaped densities that remain on other DMTs in the mbo2 mutant.The FAP58 and FAP189 subunits may also partially compensate for one another in different mutants such as mbo1 where FAP189 appears to be elevated, similar to that proposed for different paralogues of the FAP57/FAP337 complex in fap57 mutants (Lin et al., 2019).Studies of a fap189 mutant in Chlamydomonas will be required to resolve these questions.
To test the hypothesis that MBO2 and FAP58 form a heterodimer, we aligned the polypeptide sequences for MBO2, FAP58, and FAP189 using Clustal W (Supplemental Figure S4).We then used AlphaFold2 (Mirdita et al., 2022) to predict the structure of a potential MBO2/FAP58 heterodimer using full length sequences.The resulting prediction shows extensive coiled-coil domains separated by short, unstructured regions, and slightly longer, unstructured regions at the N-and C-termini of both subunits (Supplemental Figure S5).We placed the proposed MBO2/FAP58 heterodimer within the 96-nm repeat based on the positions of the MBO2-SNAP tags detected by streptavidin gold labeling (Figures 7 and 8).Because the N-terminal region of MBO2 is thought to be disordered, we could not follow the filamentous structure completely to a site near the IDA b tail domain that is predicted to be the N-terminus of MBO2 Clipping the isosurface rendering reveals the interactions of the L-shaped structure with the axoneme ruler (gold filamentous structure) located between protofilaments A02 and A03.The L-shaped structure extends upward near the base of IDA b (green) and the MIA complex (orange) and then turns distally, running below the FAP57 associated structure (blue) and N-DRC (yellow) and beyond RS2 and RS3S (unshaded) into the next 96-nm repeat, where it fades from view just below the I1 dynein.(F) A model for the proposed location of a MBO2/FAP58 heterodimer within the 96-nm repeat.AlphaFold2 was used to predict the structure of a MBO2/FAP58 heterodimer based on the Clustal W alignment of the MBO2, FAP58, and FAP189 polypeptide sequences (Supplemental Figure S6).The MBO2 structure was placed in the 96-nm repeat based on locations of the SNAP tags detected by streptavidin gold labeling (Figures 7 and 8).The density map of the DMT was obtained by classification averaging, and the class with intact IDA b structures is shown here.Because the N-terminal region of MBO2 is disordered, the filamentous structure shown in D and E could not be followed to the site predicted for N-terminal SNAP tag of MBO2.This flexible region (∼5.5 nm) is therefore indicated by a series of red dots.The proposed location for the MBO2/ FAP58 heterodimer also agrees with the positioning of a FAP189 homodimer based on mapping residues 689-719 near the MIA complex and residues 384-480 near the FAP78 distal protrusion (Walton et al., 2023).The AlphaFold2 models for the MIA complex and IDA b were added to illustrate how they are proposed to interact with the MBO2/FAP58 heterodimer (Yamamoto et al., 2013;Walton et al., 2023).Scale bars in (A) and (D) are 25 nm.
(Figure 9), and so this region is represented by a series of red dots (Figure 9F).However, the positions of the M-SNAP tag at leucine 569 and the C-SNAP tag at isoleucine 920 are consistent with the lengths of the predicted coiled-coil domains (Figure 9F).The placement of the MBO2/FAP58 heterodimer is also consistent with the mapped positions of short segments of amino acids that are conserved between FAP189 and FAP58 (See Figure Legend 7 and Walton et al., 2023) and recent chemical cross-linking experiments suggesting the presence of a CCDC146/CCDC147 heterodimer in Tetrahymena axonemes (McCafferty et al., 2023).
FAP189 has been associated with subunits of the MIA complex and FAP57 complex based on chemical cross-linking and immunoprecipitation experiments (Yamamoto et al., 2013).Given the sequence homology between FAP189 and FAP58 (∼80% sequence identity), it is likely the MBO2/FAP58 heterodimer also interacts with the MIA complex and the FAP57 complex (Figure 9E; Supplemental Figure S6), consistent with the proximity of these structures observed by cryo-ET.In Chlamydomonas, fap57 mutants are associated with reductions in FAP57 and increases in two FAP57 paralogues (FBB7 and FAP331).The fap57 mutants also display DMT-specific defects in assembly of IDA g and d.Increases in the levels of the FAP57 paralogues could partially compensate for the loss of FAP57 on certain DMTs in fap57 mutants (Lin et al., 2019).Interestingly, we have not seen any evidence that the mbo mutants disrupt the assembly of the MIA complex or FAP57 (Supplemental Table S4), even though we detected reductions in FAP331 and FAP337 in the mbo strains (Tables 1-3; Supplemental Table S4).Studies in Tetrahymena using chemical cross-linking, single particle cryo-EM and AlphaFold2 modeling have recently localized FAP337 to a "staple" formed by the coiled-coil heterodimer of CCDC96/CCDC113 (FAP184/ FAP263) that is located distal to the N-DRC (Bazan et al., 2021;Ghanaeian et al., 2023).FAP337 also interacts with the base of IDA d and the coiled-coil domains of two FAP57 paralogues, CFAP57A/CFAP57C, and CFAP57A/C interacts in turn with bases of IDA d and g (Ghanaeian et al., 2023).Collectively these observations illustrate how FAP57 and FAP337 (and their paralogues) could interconnect the MIA complex, the N-DRC, CCDC96/CCDC113, and stabilize the binding of IDA d and g (Lin et al., 2019;Bazan et al., 2021;Ghanaeian et al., 2023).In Tetrahymena, the CFAP57 paralogues also interact with an unidentified L-shaped, coiled-coil structure like the MBO2/FAP58/ FAP189 structure described above (Ghanaeian et al., 2023; Volume 35 May 1, 2024 MBO2/FAP58 stabilizes IDA b assembly | 19 Walton et al., 2023).Our observations that FAP331 and FAP337 are reduced in mbo mutants are consistent with an interaction between these FAP57-associated proteins and the MBO2-associated L-shaped structure.We have not identified any defects on the assembly of IDA d (DHC2) and IDA g (DHC3, DHC7) in the mbo strains, but impact of any reductions the FAP57-associated proteins may be offset by the WT levels of FAP57 and numerous connections between the other structures in this region.We propose that the FAP57 paralogues are located in a similar location as FAP57 and that they serve a partially redundant and possibly DMT-specific function in Chlamydomonas.The proposed location of FAP57 and its paralogues relative to other structures in the 96-nm repeat is illustrated in Supplemental Figure S6.

Interactions of the MBO2 complex with other structures in the axoneme and implications for motility
Previous work revealed that generation of ciliary and flagellar waveforms requires an asymmetric distribution of force generated by dynein arm activity along the cilia (Lin and Nicastro, 2018).MBO2 and its associated structures demonstrate that cilia have intrinsic asymmetry built into the structure of the axoneme, as well as their interconnections to multiple regulatory complexes, to facilitate both mechanical and chemical feedback control on dynein arm activity.A biophysical study of the beating patterns in Chlamydomonas, including the mbo2 mutant, showed that the asymmetric ciliary beating waveform of Chlamydomonas WT could be mathematically separated into static and dynamic components (Geyer et al., 2016;Sartori et al., 2016).That study concluded that mbo2 lacks the static component and further predicted that there were defects in IDAs in the mbo2 mutant (Geyer et al., 2016;Sartori et al., 2016).Our findings strongly suggest that MBO2 and its associated IDA b structures contribute to the generation of the static component of the ciliary waveform in Chlamydomonas.

Culture conditions, genetic analyses, and strain construction
Strains used in this study (Supplemental Table S1) were maintained on Tris-acetate phosphate (TAP) medium, but occasionally resuspended in liquid minimal medium or 10 mM HEPES, pH 7.6, to facilitate flagellar assembly and mating.Transformants were selected by cotransformation with pSI103 (encoding the aphVIII gene; Sizova et al., 2001) or pHyg2 (encoding the aphVII gene; Berthold et al., 2002) and plating on media containing 10 μg/ml paromomycin or hygromycin B.

Epitope tagging of MBO2 and characterization of candidate dhc5 mutations
Purification of genomic and plasmid DNA, restriction enzyme digests, agarose gels, and PCR reactions, were performed as previously described (Lin et al., 2019).All primers used for SNAP tagging of MBO2 and characterizing insertions into the DHC5 gene are listed in Supplemental Table S2.The plasmid containing the wildtype MBO2 gene tagged with a 2HA tag was previously described (Tam and Lefebvre, 2002), and the predicted polypeptide sequence is shown in Supplemental Figure S1.To make a construct encoding a SNAP tag at the N-terminus of MBO2, a 2033 bp gene fragment spanning two ApaI sites and encoding a codon optimized SNAP tag and first three exons and introns of MBO2 was synthesized and cloned into pUC57 (Genewiz, Azenta Life Sciences, South Plainfield, NJ).This fragment was amplified by PCR with primers spanning the ApaI sites and complementary to MBO2.After gel purification, the SNAP-tagged fragment was assembled into a ApaI digested MBO2-HA plasmid using NEBuilder (New England Biolabs, Ipswich, MA).The final N-SNAP-MBO2-HA construct encodes an MBO2 polypeptide with a SNAP tag at its N-terminus and a 2-HA tag located between amino acids 885 and 886 of the original MBO2 sequence (Supplemental Figure S1).
To insert a SNAP tag near the middle of MBO2, a 1839 bp region between two KpnI sites was amplified by PCR and subcloned into pGEM-T Easy (Promega, Madison, WI).This subclone was subjected to site-directed mutagenesis using the Q5 site-directed mutagenesis kit (New England Biolabs) to create a new HindIII site at the position encoding amino acid 569.A SNAP tag was amplified from a codonoptimized SNAP plasmid (Song et al., 2015) using primers containing HindIII sites and complementary to MBO2, gel purified, and assembled into the HindIII site of the KpnI subclone using NEBuilder.The tagged KpnI subclone was then amplified with primers containing KpnI sites, gel purified, and assembled into a KpnI-digested MBO2-HA plasmid.The final MBO2-M-SNAP-HA construct encodes an MBO2 polypeptide with a SNAP tag located between amino acids 569 and 570 and a 2-HA tag between amino acids 885 and 886 of the original MBO2 sequence (Supplemental Figure S1).
To tag the C-terminus of MBO2, a 1018 gene fragment spanning two HpaI sites and containing the last exon of MBO2 and codon optimized SNAP tag before the stop codon was synthesized and cloned into pUC57 (Genewiz).This fragment was then amplified using primers containing HpaI sites, gel purified, and assembled into the HpaI digested MBO2-HA plasmid.The final MBO2-C-SNAP construct encodes an MBO2 polypeptide with a SNAP tag at its C-terminus but lacking the HA tag (Supplemental Figure S1).

Phase contrast microscopy and measurements of swimming velocity
Motility phenotypes were assessed by phase contrast microscopy using a 20x or 40x objective on a Zeiss Axioskop microscope.Measurements of swimming velocities were made from recordings using a Rolera-MGi EM-CCD camera (Q-imaging, Surrey, BC, Canada) and the Metamorph software (Molecular Devices, San Jose, CA;VanderWaal et al., 2011;Bower et al., 2013Bower et al., , 2018;;Reck et al., 2016).At least three independent experiments were performed for each strain.Data are presented as the mean ± SD using the student's t test.Images of forward movement were obtained by collecting 1 s exposures of cells imaged with the 20x objective.Selected images were cropped, rotated, and labeled in Image J and Adobe Photoshop (San Jose, CA).
Because DHCs vary widely in abundance, purified axonemes were also fractionated by SDS-PAGE, stained briefly with Coomassie blue, and the DHC region was excised from the gel to improve the signal to noise.Following extraction and trypsin digestion, three to five replicates per sample were analyzed by MS/MS, and both the total number of peptides and total number of assigned spectra per HC isoform were determined.The relative abundance of each DHC was estimated by spectral counting (Zhu et al., 2010) and expressed as a percentage of the total spectra identified for the 1-alpha and 1-beta DHCs of the I1 dynein as previously described (Bower et al., 2013(Bower et al., , 2018)).The DHCs were also identified and quantified using the SEQUEST algorithm (Eng et al., 1994) and Proteome Discover 2.3 (Thermo Fisher Scientific).For internal calibration of the peptide masses, the recalibration node was used with a 20 ppm mass tolerance and carbamidomethyl cysteine as a fixed modification.For protein identification, we used the Chlamydomonas reinhardtii v5.6 protein FASTA database concatenated with a common lab contaminant database (www.thegpm.org/crap/)and the following SEQUEST search parameters: semitrypsin, two missed cleavage sites, minimum peptide length six, precursor mass tolerance 12 ppm, fragment mass tolerance 0.1 Da, dynamic modifications: oxidation of methionine, deamidation of asparagine and glutamine and pyro-glutamic acid modification of N-terminal glutamine, and carbamidomethyl cysteine as a fixed modification.We used the Percolator algorithm with a concatenated target-decoy database approach to control the false discovery rate (FDR; Brosch et al., 2009;Spivak et al., 2009).Each sample was analyzed in triplicate and quantified using the label free quantification workflow that includes steps for feature extraction, chromatographic alignment, peptide mapping to features, protein abundance calculation, normalization, protein relative abundance ratio calculation and hypothesis testing for significance of relative fold change.For each file, we applied the untargeted Minora Feature Detector algorithm, which is similar to the match between runs setting in MaxLFQ (Cox et al., 2014).Peptides were mapped to retention time-aligned consensus features across samples with the requirement that at least one sample contains a peptide spectral match.For each sample, protein abundances were calculated by summing abundances of consensus features for related peptides.We normalized protein abundances for each sample using two proteins: DHC1 (178 distinct peptide sequences) and DHC10 (152 distinct peptide sequences).We set the hypothesis test method to ANOVA and report p values that were adjusted using the Benjamini-Hochberg correction for FDR.
Preparation of samples for iTRAQ or TMT labeling and MS/MS analysis of whole axonemes iTRAQ labeling: Axonemes were washed in 10 mM HEPES pH 7.4 to remove salt, DTT, and protease inhibitors, then resuspended in 0.5 M triethylammonium bicarbonate pH 8.5 and processed for trypsin digestion and iTRAQ labeling as described in detail (Bower et al., 2013(Bower et al., , 2018;;Reck et al., 2016).Duplicate aliquots of axonemes (50-60 μg each) from each strain were reacted with four-plex iTRAQ reagents (114-117, AB Sciex, Foster City, CA) to obtain two technical replicates per biological sample.The four labeled aliquots were mixed and processed to remove excess trypsin, unreacted iTRAQ reagents, and buffer.The combined sample (containing two control aliquots with different iTRAQ labels and two mutant aliquots with different iTRAQ labels) was fractionated offline using high pH, C18 reversed phase chromatography (Reck et al., 2016).Approximately 500 ng of each peptide fraction was analyzed by LC-MS on a Velos Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham MA).Online capillary LC, MS/MS, database searching, and protein identification were performed as previously described (Lin-Moshier et al., 2013;Reck et al., 2016) using ProteinPilot software version 4.5 or 5.0 (AB Sciex, Foster City, CA) and version 5.5 of the Chlamydomonas genome database (https://phytozome.jgi.doe.gov/pz/portal.html).The bias factors for all samples were normalized to alpha and beta tubulin (Reck et al. 2016).The relative amount of protein in each aliquot was compared with that present in the control aliquot to obtain a protein ratio.The WT/WT or HA/HA ratios indicated the variability in labeling and protein loading between technical replicates of the same sample (typically less than 10% for all proteins).Two iTRAQ experiments with independent biological replicates were performed for mbo1, mbo2, and pf12.Between 689 and 919 proteins were identified at a 1% FDR in each experiment.The protein lists were filtered using a minimum of six peptides per protein, and those proteins that were significantly reduced (P < 0.05) in all samples using Benjamini-Hochberg correction were further analyzed.We also reanalyzed proteomics data from other iTRAQ experiments using two biological replicates of ida8 axonemes with defects in the FAP57 complex (Lin et al., 2019), and two biological replicates of pf9-2; pf28 axonemes, a double mutant that lacks ODAs and I1 dynein and assembles short (∼2.9 μm) flagella (Porter et al., 1992;Hwang et al., 2024).
TMT labeling: The proteins altered in mbo2 axonemes were analyzed in a third experiment using three independent biological replicates of WT, mbo2, and MBO2-HA axonemes and TMT16 plex labeling.We also analyzed axonemes from WT and a pf12; fap 20 mutant in another TMT experiment.Samples were digested and labeled with TMT reagents, dried down, and then fractionated offline as described above.Approximately ∼300-600 nanograms of each fraction was analyzed by capillary LC-MS with a Dionex UltiMate 3000 RSLC nano system on-line with an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) with FAIMS (high-field asymmetric waveform ion mobility) separation as described by Weise et al., 2023 with minor modifications.The LC profile was 5 to 8% solvent B at 2.5 min, 21% B at 135 min, 34% B at 180 min and 90% B at 182 min with a flowrate of 315 nl/min, where solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in ACN.The MS2 settings were: 0.7 Da quadrupole isolation window, 38% fixed collision energy, Orbitrap detection with 50K resolution at 200 m/z, first mass fixed at 110 m/z, 150 msec max injection time, 250% (1.25 × 10E5) AGC, 45 s dynamic exclusion duration with ± 10 ppm mass tolerance and exclusion lists were not shared among compensation voltages.
The peptide output from the tandem MS was processed using SEQUEST algorithm in Proteome Discoverer 2.5.The Chlamydomonas reinhardtii protein sequence database was downloaded from https://phytozome-next.jgi.doe.gov/info/Creinhardtii_v5.6 and merged with two additional sequences, DRC1 and FAP58, plus a common lab contaminant protein database (www.thegpm.org/cRAP/index.html)(16,631 total protein sequences).The database search parameters were as previously described (Weise et al., 2023) with the exception that the fragment ion tolerance was 0.08 Da.Peptides and proteins were identified using a 1% FDR and the Percolator algorithm (Käll et al., 2008) in Proteome Discover and quantified as described in Weise et al., 2023, with the exception that normalization was performed using alpha and beta tubulin.The data were also reanalyzed with the latest version of the Chlamydomonas sequence database (JGI v6.1, 16883 protein coding genes).FAP58 was annotated in early versions of the genome as Cre13.g584300,dropped for unknown reasons in v5.6, and renumbered as Cre13.g801543 in v6.1.We have included both JGI genome ID numbers here.The software used for analysis of TMT labeled samples differs in several ways from the Protein Pilot software used for analysis of iTRAQ labeled samples, but both methods provide estimates of relative protein ratios based on the quantification of isobaric tags.
Cryo-sample preparation, cryo-electron tomography, and image processing The purified axoneme pellet was resuspended in HMEEK buffer (30 mM HEPES, pH7.4, 5 mM MgSO 4 , 1 mM EGTA, 0.1 mM EDTA, 25 mM KCl), and the suspension was directly used for cryo-sample preparation.For visualization of the SNAP tags, streptavidin nanogold labeling was performed on axonemes from strains rescued with SNAP-tagged versions of the MBO2 as previously described (Song et al., 2015).Briefly, 1 µl of 1 mM BG-(PEG)12-biotin (New England Biolabs; PEG linker available on request) was added to 200 µl of axonemes.A control sample was also prepared without added BG-(PEG)12-biotin.Both suspensions were incubated overnight at 4°C, followed by three cycles of resuspension with HMEEK buffer and centrifugation at 10,000 g for 10 min at 4°C.The axoneme pellets were resuspended in 200 µl of buffer, and then either 5 µl of 1.4-nm-sized streptavidin nanogold particles (strep-Au, Nanoprobes) or 5 µl of buffer were added, and the two suspensions were incubated at 4°C in the dark for 3 h with rotation.The samples were then diluted with 1 ml of HMEEK buffer, pelleted by centrifugation at 10,000 g for 10 min at 4°C, carefully resuspended in 200 µl of HMEEK buffer, and used for cryo-sample preparation.
Cryo-sample preparation, cryo-ET and image processing were done as previously described (Nicastro et al., 2006;Nicastro, 2009;Heuser et al., 2009;Lin et al., 2014).Briefly, Quantifoil copper grids (Quantifoil Micro Tools, Jena, Germany) with a holey carbon film (R2/2, 200 mesh) were glow discharged for 30 s at -40 mA and loaded with 3 µl of axoneme sample and 1 µl of BSA-coated, fivefold concentrated 10-nm colloidal gold (Sigma-Aldrich, St. Louis, MO; Iancu et al., 2006).After brief mixing, grids were blotted from the back side with filter paper (Whatman #1) for 1.5-3 s and plunge frozen in liquid ethane using a home-made plunger.Vitrified samples were either loaded onto a cryo holder (Gatan, Pleasanton, CA) and transferred to a Tecnai F30 or assembled into autogrids and transferred to a Titan Krios transmission electron microscope (Thermo Fisher Scientific, Waltham, MA) for imaging.Single-axis tilt series of noncompressed, intact axonemes were acquired using the software package SerialEM (Mastronarde, 2005).Typically, 50 to 70 images were recorded for each tilt series while the specimen was tilted from about -65 to +65° in 1.5 to 2.5° increments.A dose-symmetric scheme was applied for data collection (Hagen et al., 2017).The magnification was set to 13,500X (∼1 nm effective pixel size) with -6 to -8 µm defocus (for Tecnai F30) or 26,000X (∼0.55 nm effective pixel size) with -0.5 µm defocus using a Volta phase plate (Danev et al., 2014) (for Titan Krios).The microscope was operated in low dose mode at 300 keV and the accumulative electron dose of the sample was restricted to ∼100 e/Å 2 to minimize radiation damage.Electron micrographs were recorded with a 2k x 2k CCD camera (on Tecnai F30) or with a 4k x 4k K2 electron direct camera.The K2 camera was operated in counting mode (0.4-s exposure time per frame and 15 frames per tilt series with a dose rate of eight e -/p/s.Both TEMs were equipped with a postcolumn energy filter (Gatan, Pleasanton, CA) that was operated in zero-loss mode with a slit width of 20 eV.
The raw frames from the K2 camera were aligned for motioncorrection with a script from the IMOD software (Kremer et al., 1996).Three-dimensional tomograms were reconstructed using fiducial alignment of the tilt series images and weighted backprojection using IMOD.Subtomograms containing the 96-nm repeat units were further aligned and averaged using PEET (Nicastro et al., 2006), resulting in averaged three-dimensional structures with compensated missing wedge effect, reduced noise, and thus increased resolution.For doublet-specific averaging, the nine outer DMTs were identified based on DMT-specific features (Bui et al., 2012;Lin et al., 2012), and repeats from individual DMTs were averaged.To further analyze structural defects that appeared heterogeneous or to identify the sites labeled with nanogold particles, classification analyses were performed on the aligned sub-tomograms using the PEET program (Heumann et al., 2011).Appropriate masks were applied to focus the classification analysis on specific regions of interest, and sub-tomograms containing the same structures were grouped into class averages.The structures were mapped onto their respective locations in the raw tomograms to determine the distribution of the different classes.The numbers of tomograms and sub-tomograms that were analyzed and the resolutions of the resulting averages are summarized in Supplemental Table S5.The resolution was estimated at the center of the sub-tomogram volume using the Fourier shell correlation method with a criterion of 0.5.The structures were visualized as two-dimensional tomographic slices and three-dimensional isosurface renderings using IMOD and UCSF Chimera (Pettersen et al., 2004), respectively.The structure of the MBO2/FAP58 heterodimer was predicted using the AlphaFold 2 software (Jumper et al., 2021;Mirdita et al., 2022).To place the pseudoatomic model into the L-shaped structure, the two longest coiled-coil regions were arranged into one long filament and the shorter coiled-coil tilted into an about 90° angle relative to the filament (Figure 7F).

FIGURE 1 :
FIGURE 1: Schematic diagram of structures in the 96-nm axoneme repeat.(A) Drawing of the biflagellate green alga Chlamydomonas and diagram of a cross-section through the axoneme showing the nine outer DMTs surrounding the inner CPC.(B) Diagram of a single DMT shown in cross-section at higher magnification with the complete A-tubule (At) and associated B-tubule(Bt).The inner junction between the At and Bt is composed of alternating subunits of PACRG and FAP20 (aqua).The A-tubule contains a multisubunit ODA with three motor domains (lilac), multiple IDA isoforms (pink), the NDRC (yellow), and one of three radial spokes (RS, grey).The small light blue dot indicates the location of the ruler subunits FAP59 (CCDC39) and FAP172 (CCDC40) that determine the spacing of the RSs and IDAs.Also shown in cross-section is a portion of the FAP57 complex (dark blue).(C) Diagram of a single DMT shown in longitudinal view from the proximal (pro) to distal (dist) region of the 96-nm repeat, containing four ODAs (lilac), the seven IDA isoforms I1 (f), a, b, c, e, g, d (pink), and the three RS structures (RS1, RS2, RS3S), with I1 (f) above RS1 and the N-DRC (yellow) above RS2.The two I1 motor domains (α, β) are also attached to the DMT through the Tether-Tether Head (T/TH) (fuschia).The MIA complex (brown) links the base of I1 to the ODAs, the DMT, and the proximal portion of the FAP57 complex (dark blue).The coiled-coil domains of the FAP57 extend beyond the N-DRC to contact IDA g and d.

FIGURE 2 :
FIGURE 2: MBO2 is involved in stabilizing the assembly of DHC5.(A) A schematic diagram of the MBO2 polypeptide showing the location of coiled-coil domains (black) and a disordered region in the C-terminus gray).Also shown is the position of the HA epitope tag.(B) Transformation of a mbo2 mutant with MBO2-HA restores forward swimming.The forward swimming velocities of WT, mbo2, and HA-rescued strains (HA1-HA4) were measured by phase contrast light microscopy.All the HA-rescued strains swam forwards significantly faster (P < 0.5) than mbo2, but significantly slower than WT, consistent with previous reports (Tam and Lefebvre, 2002).(C) Western blots of axonemes probed with different antibodies show the recovery of MBO2 and DHC5 in the HA-rescued strains.DHC9 and DIC2 are loading controls for other IDA and ODA isoforms.(D) Western blots of mbo1 and mbo3 axonemes show that MBO2 and DHC5 are also reduced in these two strains.(E) WT and mbo2; MBO2-HA axonemes were subjected to sequential extraction with 0.6 M NaCl and 0.2-0.6 M NaI buffers, and analyzed on Western blots (WA, whole axoneme; OD, extracted outer doublets).The majority of MBO2 was extracted with 0.4 M NaI, whereas dynein subunits (DIC2) were largely extracted with 0.6 M NaCl.(F) Western blot of WT axonemes (A) that were extracted with increasing concentrations of Sarkosyl (S, supernatant; P, pellet).(G) Western blot of axonemes from different motility mutants lacking outer arms (pf28), inner arms (ida2, ida4, ida5), B-tubule beaks (pf12, mbo2), and radial spokes (pf14).

FIGURE 3 :
FIGURE 3: IDA b is reduced in the 96 nm axonemal repeat from mbo2.(A-H) Tomographic slices (A-C, E-G) and isosurface renderings (D and H) of the averaged 96-nm repeats from all DMTs of WT (A-D) and mbo2 (E-H) axonemes.Blue lines in (A and E) indicate the locations of the slices in the respective panels.IDA b can be visualized in WT (red arrowheads) but not in mbo2 (white arrowheads).(I) The presence (+) and absence (-) of IDA b in the 96-nm repeats from the proximal and medial-distal regions of WT and mbo2 axonemes.(J) The bar graph shows the fraction of repeats with IDA b for each DMT from WT (green) and mbo2 (light green) in the proximal and distal regions.(K) Distribution pattern of IDA b in four tomograms of proximal and distal regions from WT (left) and mbo2 (right).Each grid represents a single repeat, and the colors indicate whether IDA b is present (green) or absent (white) in the repeat.From bottom to top, the grids represent the repeats in the proximal-to-distal direction (gray arrow).Note that IDA b is not present in the proximal region and Tomo 1 of WT shows a transition from the proximal-to-distal region.Other labels: 1α, β, the I1 dynein α-and β-head; a-g, single-headed IDAs; ICLC, intermediate chain, and light chain complex of I1 dynein; IDA, inner dynein arm; N-DRC; ODA, outer dynein arm; RS1/2/3S, radial spokes one, two, and three stand-in.Scale bar in G, 20 nm (valid for A-C and E-G).

FIGURE 4 :
FIGURE 4: DMT specific defects in mbo2.(A and B) Bottom view of the three-dimensional isosurface renderings of the averaged 96-nm repeats from all DMTs in WT (A) and mbo2 (B) axonemes.Radial spokes (RS) were cropped to allow an unhindered view of the IDA-to-DMT-docking region.Motor domain of IDA b is colored green (C-K) Comparison of the IDA b tail-associated structures across nine DMTs between WT (left) and mbo2 (right) focusing on the region marked by the dashed box in A. Based on the structural characterizations in WT and defects in mbo2, the WT DMTs were categorized into three groups: DMTs1, nine (blue), DMTs 2-4 (purple) and DMTs 5-8 (red).The black arrowhead in WT DMT5 indicates a DMT5-specific structure connecting RS1 and RS2.(L) A schematic drawing of the axoneme in cross-section to show the asymmetric distribution of the IDA b associated structures on the three classes of DMTs.

FIGURE 5 :
FIGURE 5: The ciliary axoneme contains several DMT specific structures.(A-F) The B-tubule beak structures are found in DMTs 1, 5, and 6 of WT axonemes but missing in DMT5 of mbo2 axonemes.Tomographic slices (left two columns viewed in cross-sectional and longitudinal orientations) and three-dimensional isosurface renderings (viewed in longitudinal orientation) show the B-tubule beak structure in DMT1 (A and B), DMT5 (C and D), and DMT6 (E and F) of WT and mbo2 axonemes.The blue line in (A) indicates the location of the longitudinal slices.Cyan arrowheads indicate presence of the beak structure, which is missing in DMT5 in mbo2 (D, white arrowheads).(G) Tomographic slice (left) and three-dimensional isosurface renderings (right) of the averaged WT DMT structure viewed from cross-sectional (the left two columns) and bottom-up (the 3 rd column) orientations.The density corresponding to the MBO2 protein and its associated MIA, FAP57 protein are indicated.(H-J) Tomographic slices (top row) and three-dimensional isosurface renderings (bottom row) showing three representative DMT specific structures that are located near the L-shaped MBO2 filament.Note that the orientations of the tomographic slices are indicated by the blue lines in (G).In (H), a density extending from the C-terminal region of MBO2 was only observed on DMT2 (magenta arrowheads) but not on other DMTs (white arrowheads).In (I), a structure that attaches near the base of the IDA b tail domain was identified only in the proximal region of DMT5 (magenta arrowheads).This density was not seen in the distal region of DMT5 (white arrowheads) or on other DMTs.In (J), an extra density protruding from the FAP57 complex was only found on DMT9 (magenta arrowheads) but not on other DMTs (white arrowheads).Scale bars in (A) and (G) are 20 nm and valid for the EM images in (A-F) and (G-J), respectively.

FIGURE 6 :
FIGURE 6: Rescue of biochemical, motility, and structural defects in the SNAP-tagged MBO2 strains.(A) Schematic diagram of MBO2 showing the insertion of SNAP tags at the N-terminus (N-SNAP, M1), in the middle (M-SNAP, L569) and at the C-terminus (C-SNAP, I920) of the polypeptide.Also shown are the coiled-coil domains (black), disordered region (gray) and HA tag.(B) Western blot of axonemes from WT, mbo2, and MBO2::SNAP rescued strains (N, M, and C) probed with different antibodies.Both DHC5 and MBO2 are reassembled in the rescued strains; DRC1 is a loading control.(C) Transformation of mbo2 with each construct significantly increased (P < 0.05) swimming velocity relative to mbo2 (see asterisks).N-SNAP and C-SNAP were significantly faster than M-SNAP, slower than WT, but not significantly different from one another.(D) Forward swimming trajectories of each strain are shown here for an interval of 1 s.(The mbo2 strain is swimming backwards.)Scale bar, 50 mm.(E) Percentage of 96-nm axoneme repeats that contain IDA b in WT, mbo2, mbo2; MBO2::SNAP (N), mbo2; SNAP::MBO2 (M) and mbo2; MBO2::SNAP (C) strains.The assembly of IDA b is increased in the rescued strains as compared with mbo2.(F) Bar graph shows the percentage of 96-nm repeats that contain IDA b for each DMT from WT, the three SNAP-tagged rescued strains, and mbo2.IDA b is increased on DMTs 2-8 in all rescued strains as compared with mbo2.(G) SDS-PAGE of SNAP-tagged axonemes labeled with streptavidin gold and detected by silver enhancement.The yellow arrows mark the SNAP-tagged MBO2 polypeptides.Tubulin was stained with Coomassie Brilliant Blue (CBB) as a loading control.(H) Percentage of 96-nm repeats with gold-labeled SNAP-tags found on each DMT in mbo2; MBO2::SNAP (C), mbo2; SNAP::MBO2 (N) and mbo2; MBO2::SNAP (M).

FIGURE 7 :
FIGURE 7: Localization of the N-terminus, middle region, and C-terminus of the MBO2 polypeptide in the 96-nm axoneme repeat.The locations of the three different SNAP tags were revealed by comparing WT (A, C, and E) with streptavidin-gold labeling of different SNAPtagged rescued strains (B, D, and F) and class averaging of the axonemal repeats with tagdensity.Shown are tomographic slices (left two columns) and three-dimensional isosurface renderings (third column).(A and B) The region containing the structures associated with the tail domain of IDA b are viewed in cross-sectional and bottom orientations in WT (A) and mbo2; N-SNAP::MBO2 rescued axonemes (B).The blue line in (A) indicates the location of the slices viewed in the bottom orientations.The L-shaped, MBO2-associated density of one 96-nm repeat is colored in red in the three-dimensional isosurface renderings (A-F).Classification analysis revealed an additional density near the tail domain of IDA b in ∼20% of the repeats (indicated by yellow arrowheads in B).WT repeats lack similar density in this position (white arrowheads in A). (C and D) Tomographic slices viewed in cross-sectional and front orientations and cropped three-dimensional isosurface renderings viewed in front orientation of the averaged repeats from WT (C) and mbo2; MBO2::M-SNAP rescued axonemes (D).The blue line in (C) indicates the location of the slice viewed in front orientation.Classification analysis of mbo2; MBO2::SNAP (M) averages revealed an additional density (yellow arrowheads in D) in ∼15% of the repeats; this density is located on the surface of the A-tubule between protofilaments A04 and A05.A similar density in this was not observed by classification of the WT repeats (white arrowhead in C). (E and F) Tomographic slices and three-dimensional isosurface renderings viewed in cross and bottom orientations from WT (E) and mbo2; MBO2::C-SNAP (F) axonemes in the region close to the surface of the DMT below IDA b.The blue line in (E) indicates the locations of the slices viewed in the bottom orientations in (E) and (F).Classification analysis of the mbo2; MBO::C-SNAP rescued axonemes revealed an additional density (yellow arrowheads in F) in ∼31% of the repeats.This density indicates the likely location of the C-terminus of MBO2 close to the inner junction.A similar density was not seen by classification of WT axonemes (white arrowheads in E).Other labels: A t /B t , A-and B-tubule; a-g, single-headed IDAs; ODA, outer dynein arm; RS, radial spoke.Scale bars in A, C, and E, 20 nm (valid for all EM images).

FIGURE 8 :
FIGURE 8: Doublet specific asymmetry of IDA b tail-associated structures.(A and B) threedimensional isosurface renderings of the 96-nm repeats obtained by averaging DMTs 3-4 (A) and DMTs 5-8 (B) of WT axonemes.The regions marked by dashed boxes on the images in the top row were rotated and enlarged in the images shown in the bottom row for better visualization of the structures associated with the IDA b tail domain.These structures are highlighted by purple and red colors in DMTs 3-4 and DMTs 5-8, respectively.Locations of the additional densities seen by gold labeling of the C-terminal, N-terminal, and Mid-region SNAP tags in MBO2 are indicated by the yellow dots.(C) Schematic drawings of the cross-section of WT (left) and mbo2 (right) axonemes showing the arrangement of MBO2 and IDA b tailassociated structures across the nine DMTs.Our data suggest that in WT, MBO2 is missing on both DMT1 and nine, because no defects were observed on these DMTs in the mbo2 mutant.The distinct variations observed in the IDA b tail-associated structures on different DMTs are indicated by different colors (green, light green, white).The structure is unchanged between WT and mbo2 on DMT1 or DMT9 (see blue density) and only slightly affected on DMT2 (purple to magenta).Loss of MBO2 causes the IDA b tail-associated structures to be completely missing on DMT5 -DMT8 (white densities) and alters their morphology on DMT3 -DMT4 (from purple to blue), resembling the structures seen on DMT1 and DMT9.Other labels: At/Bt, A-and B-tubule; a-g, single-headed IDAs; 1α, 1β, the I1 dynein α-and β-head; IJ, inner junction; N-DRC; ODA, outer dynein arm; RS1, RS2, radial spoke 1 and 2.

FIGURE 9 :
FIGURE 9: Summary images illustrating the proposed location of the MBO2/FAP58 heterodimer and its interactions with other regulatory complexes in the 96-nm repeat.(A and B) Tomographic slice and three-dimensional isosurface rendering of the DMT in crosssection in the region between RS1 and RS2 indicating a filamentous structure on the surface of DMT (circled in red in A and colored in red in B) running from the A02 protofilament to the cleft between protofilaments A04 and A05.(C) Rotating the isosurface model 90° along y-axis shows the position of the red structure below IDA b (in green) and the globular domain of FAP57 (in blue).The red structure continues along the surface of the DMT, running behind the N-DRC (yellow) and extending into the next 96-nm repeat.(D) A tomographic slice taken through the DMT along the plane illustrated by the line in (A) and (E) the corresponding isosurface model tilted 60 degrees along x-axis from the view in (C) shows the MBO2-associated, L-shaped structure running vertically from protofilament A02 to A04 and then bending and extending distally along the cleft between protofilaments A04 and A05 (indicated by red arrowheads in D and red structure in E).Clipping the isosurface rendering reveals the interactions of the L-shaped structure with the axoneme ruler (gold filamentous structure) located between protofilaments A02 and A03.The L-shaped structure extends upward near the base of IDA b(green) and the MIA complex (orange) and then turns distally, running below the FAP57 associated structure (blue) and N-DRC (yellow) and beyond RS2 and RS3S (unshaded) into the next 96-nm repeat, where it fades from view just below the I1 dynein.(F) A model for the proposed location of a MBO2/FAP58 heterodimer within the 96-nm repeat.AlphaFold2 was used to predict the structure of a MBO2/FAP58 heterodimer based on the Clustal W alignment of the MBO2, FAP58, and FAP189 polypeptide sequences (Supplemental FigureS6).The MBO2 structure was placed in the 96-nm repeat based on locations of the SNAP tags detected by streptavidin gold labeling (Figures7 and 8).The density map of the DMT was obtained by classification averaging, and the class with intact IDA b structures is shown here.Because the N-terminal region of MBO2 is disordered, the filamentous structure shown in D and E could not be followed to the site predicted for N-terminal SNAP tag of MBO2.This flexible region (∼5.5 nm) is therefore indicated by a series of red dots.The proposed location for the MBO2/ FAP58 heterodimer also agrees with the positioning of a FAP189 homodimer based on mapping residues 689-719 near the MIA complex and residues 384-480 near the FAP78 distal protrusion(Walton et al., 2023).The AlphaFold2 models for the MIA complex and IDA b were added to illustrate how they are proposed to interact with the MBO2/FAP58 heterodimer(Yamamoto et al., 2013;Walton et al., 2023).Scale bars in (A) and (D) are 25 nm.

TABLE 1 :
Motility mutants compared in this study.Continued

TABLE 2 :
Protein ratios in axonemes from mbo2 and rescued MBO2-HA strains.Continued